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. 2021 Jun 22;118(25):e2101276118.
doi: 10.1073/pnas.2101276118.

Evolution of bacterial steroid biosynthesis and its impact on eukaryogenesis

Yosuke Hoshino  1 , Eric A Gaucher  1
Affiliations

Evolution of bacterial steroid biosynthesis and its impact on eukaryogenesis

Yosuke Hoshino et al. Proc Natl Acad Sci U S A. .

Abstract

Steroids are components of the eukaryotic cellular membrane and have indispensable roles in the process of eukaryotic endocytosis by regulating membrane fluidity and permeability. In particular, steroids may have been a structural prerequisite for the acquisition of mitochondria via endocytosis during eukaryogenesis. While eukaryotes are inferred to have evolved from an archaeal lineage, there is little similarity between the eukaryotic and archaeal cellular membranes. As such, the evolution of eukaryotic cellular membranes has limited our understanding of eukaryogenesis. Despite evolving from archaea, the eukaryotic cellular membrane is essentially a fatty acid bacterial-type membrane, which implies a substantial bacterial contribution to the evolution of the eukaryotic cellular membrane. Here, we address the evolution of steroid biosynthesis in eukaryotes by combining ancestral sequence reconstruction and comprehensive phylogenetic analyses of steroid biosynthesis genes. Contrary to the traditional assumption that eukaryotic steroid biosynthesis evolved within eukaryotes, most steroid biosynthesis genes are inferred to be derived from bacteria. In particular, aerobic deltaproteobacteria (myxobacteria) seem to have mediated the transfer of key genes for steroid biosynthesis to eukaryotes. Analyses of resurrected steroid biosynthesis enzymes suggest that the steroid biosynthesis pathway in early eukaryotes may have been similar to the pathway seen in modern plants and algae. These resurrected proteins also experimentally demonstrate that molecular oxygen was required to establish the modern eukaryotic cellular membrane during eukaryogenesis. Our study provides unique insight into relationships between early eukaryotes and other bacteria in addition to the well-known endosymbiosis with alphaproteobacteria.

Keywords: biomarker; cellular membrane; eukaryogenesis; sequence reconstruction; steroids.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Distribution of steroid biosynthesis genes in select bacterial taxa and eukaryotes. Representative enzymatic products are shown at each step. The evolutionary relationship within the Deltaproteobacteria and Oligoflexia group (blue color) is shown in the inset. Underlined genes in bacteria indicate that only a small number of species possess the gene within the indicated group. Genes lightly shaded in LECA indicate uncertainty in the presence of the gene. The reaction order within Stage 2 is generally from left (cyclization) to right (modification of desaturation), but there are variations after cyclization depending on some species. The MVA pathway in green color indicates the eukaryotic type, while the pathway in red color indicates the archaeal type. ERG24 is separately treated from other desaturation-modifying genes. See SI Appendix, Fig. S1 for gene abbreviations.
Fig. 2.
Fig. 2.
Bayesian phylogenetic trees for select Stage 1 and 2 steroid biosynthesis genes. Filled black circles indicate that the node support is >75% for maximum likelihood inference and >75% for Bayesian inference for all four trees (node support is shown only for major clades). Filled gray circles indicate that the node support is above the same threshold for three out of four trees. The nodes with less than 0.5 support are collapsed. The black line indicates the outgroup. (Scale bar represents 0.5 amino acid replacements per site per unit evolutionary time.) See SI Appendix, Figs. S3–S5 for other gene trees and SI Appendix, Figs. S9–S19 for the complete trees with the species annotation. Abbreviations: CYP51, cytochrome P450 family 51 (sterol C-14 demethylase); DPMD, diphosphomevalonate kinase; OSC; PMVK, phosphomevalonate kinase; SQMO, squalene monooxygenase; and SQS.
Fig. 3.
Fig. 3.
Concatenated Bayesian phylogenetic tree of eight Stage 2 steroid biosynthesis genes (SQMO, OSC, CYP51, ERG24, ERG25, ERG26, ERG27 analog, and CPI1). Sequences are selected from species that have a potential steroid biosynthesis pathway for at least protosterols. The distribution of Stage 1 and 2 steroid biosynthesis genes is also shown for individual species. The tree topology remains nearly identical under the four different substitution models. The nodes with less than 0.5 support are collapsed. (Scale bar represents 0.4 amino acid replacements per site per unit evolutionary time.) See SI Appendix, Fig. S6 for the complete tree with the species annotation and SI Appendix, Table S3 for the source data.
Fig. 4.
Fig. 4.
Bayesian phylogenetic tree of terpene cyclase family and product profiles for three resurrected OSCs (nodes A, B, and C). The tree topology remains nearly identical under the four different substitution models. The nodes with less than 0.5 support are collapsed. (Scale bar represents 0.5 amino acid replacements per site per unit evolutionary time.) See SI Appendix, Fig. S7 for the complete tree with the species annotation.
Fig. 5.
Fig. 5.
Hypothetical steroid biosynthesis pathway for pre-LECA eukaryotes that acquired eight Stage 2 genes and, additionally, ERG2 and SMT genes (Stage 1 not displayed). Steroid biosynthesis pathways in modern eukaryotes other than Opisthokonta and Archaeplastida are largely unknown. The inferred pre-LECA pathway functionally overlaps with part of the modern steroid biosynthesis pathways for Archaeplastida and Opisthokonta (light blue and yellow colors). Since the reaction order of the pre-LECA pathway is unknown, the identity of intermediate steroid compounds is not clear and thus only possible end products are shown. The C-24 ethylation of steroids for the stigmasterol biosynthesis requires a pair of paralogous SMT genes (SMT1 and SMT2) and thus is inferred to have been not possible in both pre-LECA eukaryotes and LECA, because they had only a single copy of the gene. Abbreviations: CAS, cycloartenol synthase and LAS, lanosterol synthase. See SI Appendix, Fig. S1 for gene abbreviations.
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